Viral Vector Delivery of DREADDs for CNS Therapy

: Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are genetically modified G-protein-coupled receptors (GPCRs), which can be activated by a synthetic ligand that is otherwise inert at endogenous receptors. DREADDs can be expressed in cells in the central nervous system (CNS) and subsequently offer the opportunity for remote and reversible silencing or activation of the target cells when the synthetic ligand is systemically administered. In neuroscience, DREADDs have thus far shown to be useful tools for several areas of research. Furthermore, they offer considerable potential for use as a gene therapy strategy for neurological disorders. However, in order to design a DREADD-based gene therapy, it is necessary to first evaluate the viral vector delivery methods utilised to deliver these chemogenetic tools in the literature. This review evaluates each of the prominent strategies currently utilised for DREADD delivery, discussing their respective advantages and limitations. It focuses on Adeno-Associated Virus (AAV)- and lentivirus-based systems, and the manipulation of these through cell-type specific promoters and pseudotyping. Furthermore, we address how virally mediated DREADD delivery could be improved in order to make it a viable gene therapy strategy and thus expand its translational potential.

Insulin receptor activation by proinsulin preserves synapses and vision in retinitis pigmentosa

Background Synaptic loss, neuronal cell death, and circuit remodeling are common features of central nervous system neurodegenerative disorders. Retinitis pigmentosa, the leading cause of inherited blindness, is a group of retinal dystrophies characterized by photoreceptor cell dysfunction and death. The insulin receptor, a key controller of metabolism, also regulates neuronal survival and synaptic formation, maintenance, and activity. Indeed, deficient insulin receptor signaling has been implicated in several brain neurodegenerative pathologies. Methods We employed a gene therapy strategy to enhance insulin receptor signaling in the Pde6b rd10/rd10 mouse model of RP by using recombinant AAV serotype 2/1 viral vectors bearing cDNA from the human proinsulin gene. Insulin receptor expression and signaling were analyzed by PCR and immunostaining. Synaptic connectivity was evaluated by electron microscopy and immunostaining. Proinsulin levels were measured by ELISA. Photoreceptor preservation was assayed by histological analysis and visual function was assessed by electroretinography and optomotor test. Results We present evidence linking impaired insulin receptor signaling with retinitis pigmentosa. We found a selective decrease in the levels of the insulin receptor and its downstream effector phospho-S6 in retinal horizontal cell axons in the rd10 mouse model of retinitis pigmentosa, as well as aberrant synapses between rod photoreceptors and the postsynaptic terminals of horizontal and bipolar cells. A gene therapy strategy to induce sustained proinsulin production restored retinal insulin receptor signaling, by increasing S6 phosphorylation, without peripheral metabolic consequences. Moreover, proinsulin preserved photoreceptor synaptic connectivity and prolonged visual function as determined by electroretinography and optomotor tests. Conclusion These findings point to a disease-modifying role of insulin receptor and support the therapeutic potential of proinsulin in retinitis pigmentosa.

Deletion of M-opsin prevents “M cone” degeneration in a mouse model of Leber congenital amaurosis

Mutations in RPE65 or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal synthesis and cause Leber congenital amaurosis (LCA). In Lrat−/− mouse model, mislocalized medium (M)-wavelength sensitive opsin was degraded whereas mislocalized short (S)-wavelength sensitive opsin accumulated before the onset of cone degeneration. The mechanism for the foveal medium (M)/long (L)-wavelength-sensitive cone degeneration in LCA is unknown. By crossing Lrat−/− mice with a proteasome reporter mouse line, we showed that M-opsin enriched dorsal cones in Lrat−/− mice exhibit proteasome stress due to the degradation of large amounts of M-opsin. Deletion of M-opsin relieves the proteasome stress and completely prevents “M cone” degeneration in Lrat−/−Opn1sw−/− mice (a pure “M cone” LCA model, Opn1sw−/− encoding S-opsin) for at least 12 months. Our results suggest that M-opsin degradation associated proteasome stress plays a major role in “M cone” degeneration in Lrat−/− model. This finding may represent a general mechanism for “M cone” degeneration for multiple forms of cone degeneration due to M-opsin mislocalization and degradation. Our results have important implications for the current gene therapy strategy for LCA that emphasizes the need for a combinatorial therapy to both improve vision and slow photoreceptor degeneration.